Herpes Simplex Virus Type 1 Virus Research Articles

The anti-tumor efficacy of a recombinant oncolytic herpes simplex virus mediated CRISPR/Cas9 delivery targeting in HPV16-positive cervical cancer

Cervical cancer, often driven by high-risk human papillomavirus (HPV) infections such as HPV16 or HPV18, remains a leading cause of cancer-related deaths. HPV16, found in about 90% of cervical cancer patients, harbors key oncogenic related genes (E6, E7, E2, E5) and an upstream regulatory region (URR) that contribute to cancer progression. This study introduces a novel approach using a recombinant oncolytic herpes simplex virus type 1 (HSV-1) named SONC103, armed with a CRISPR/Cas9 gene editing system. The aim was to target and disrupt integrated HPV16 genes in cervical cancer cells. Results demonstrated SONC103's capability to specifically and effectively knock down HPV16 oncogenes, thereby reducing cell proliferation and promoting apoptosis. Analyses further revealed loss of HPV16 DNA probes in infected cells' chromosomes, significant regulation of cellular processes related to tumor apoptosis, and downregulation of E6/E7 oncoproteins while increasing tumor suppressor proteins P53 and pRB. Notably, SONC103 exhibited substantial inhibition of tumor growth in a murine xenograft cervical cancer model. This study showcases the potential of the recombinant oncolytic HSV-1 virus (SONC103) in combating HPV16-positive cervical cancer by targeting oncogenes and facilitating oncolysis.

Viral Etiology of Aseptic Meningitis and Clinical Prediction of Herpes Simplex Virus Type 2 Meningitis.

Aseptic meningitis comprises meningeal inflammation and cerebrospinal fluid (CSF) pleocytosis without positive Gram stain and culture. Regional differences exist in the prevalence of viral etiologies of aseptic meningitis. We aimed to assess the etiologies of aseptic meningitis in immunocompetent adults, focusing on herpes simplex virus type 2 (HSV-2). This study retrospectively analyzed immunocompetent adults diagnosed with meningitis at a Korean tertiary care hospital from 2016 to 2018. Aseptic meningitis was defined through clinical and CSF analysis. We compared clinical and laboratory characteristics across viral etiologies and investigated predictors of HSV-2 meningitis. A total of 98 patients (46.9% female) with aseptic meningitis were finally enrolled. The etiologies of aseptic meningitis were identified in 62 patients (63.3%), including enterovirus (28.5%), HSV-2 (16.3%), and varicella zoster virus (VZV, 15.3%). HSV-2 showed female predominance, with shorter admission times with longer hospital stays and a recurrent meningitis history. Compared to other viral etiologies, HSV-2 showed higher CSF white blood cell (WBC) counts and protein levels but lower C-reactive protein (CRP) levels. A random forest model identified previous meningitis history and serum CRP level as key predictors of HSV-2 meningitis. This study provides insights into the etiologies of aseptic meningitis in a specific Korean region, identifying HSV-2 as a notable cause. The prediction model suggested that the clinical history of previous meningitis and serum CRP level may guide clinical assessment of meningitis.

Antimicrobial Efficacy of a Vegetable Oil Plasticizer in PVC Matrices.

The growing prevalence of bacterial and viral infections, highlighted by the recent COVID-19 pandemic, urgently calls for new antimicrobial strategies. To this end, we have synthesized and characterized a novel fatty acid epoxy-ester plasticizer for polymers, named GDE. GDE is not only sustainable and user-friendly but also demonstrates superior plasticizing properties, while its epoxy components improve the heat stability of PVC-based matrices. A key feature of GDE is its ability to confer antimicrobial properties to surfaces. Indeed, upon contact, this material can effectively kill enveloped viruses, such as herpes simplex virus type 1 (HSV-1) and the β-coronavirus prototype HCoV-OC43, but it is ineffective against nonenveloped viruses like human adenovirus (HAdV). Further analysis using transmission electron microscopy (TEM) on HSV-1 virions exposed to GDE showed significant structural damage, indicating that GDE can interfere with the viral envelope, potentially causing leakage. Moreover, GDE demonstrates antibacterial activity, albeit to a lesser extent, against notorious pathogens such as Staphylococcus aureus and Escherichia coli. Overall, this newly developed plasticizer shows significant potential as an antimicrobial agent suitable for use in both community and healthcare settings to curb the spread of infections caused by microorganisms contaminating physical surfaces.

Function and Mechanism of Antiviral Wasp Venom Peptide Protopolybia-MP III and Its Derivatives against HSV-1.

Viruses are one of the leading causes of human disease, and many highly pathogenic viruses still have no specific treatment drugs. Therefore, producing new antiviral drugs is an urgent matter. In our study, we first found that the natural wasp venom peptide Protopolybia-MP III had a significant inhibitory effect on herpes simplex virus type 1 (HSV-1) replication in vitro by using quantitative real-time PCR (qPCR), Western blotting, and plaque-forming assays. Immunofluorescence analysis showed Protopolybia-MP III could enter cells, and it inhibited multiple stages of the HSV-1 life cycle, including the attachment, entry/fusion, and post-entry stages. Furthermore, ultracentrifugation and electron microscopy detected that Protopolybia-MP III significantly suppressed HSV-1 virion infectivity at different temperatures by destroying the integrity of the HSV-1 virion. Finally, by comparing the antiviral activity of Protopolybia-MP III and its mutants, a series of peptides with better anti-HSV-1 activity were identified. Overall, this work found the function and mechanism of the antiviral wasp venom peptide Protopolybia-MP III and its derivatives against HSV-1 and laid the foundation for the research and development of wasp venom-derived antiviral candidate peptide drugs.

HSV1-induced enhancement of productive HIV-1 replication is associated with interferon pathway downregulation in human macrophages.

Herpesviruses are common co-pathogens in individuals infected with human immunodeficiency virus (HIV). Herpes simplex virus type 1 (HSV1) enhances HIV-1 replication and has evolved mechanisms to evade or disrupt host innate immune responses, including interference with interferon (IFN) signalling pathways. The aimed of this work was evaluated whether it HSV1 affects HIV-1 replication through the modulation of the IFN pathway in human macrophages. Co-infections with HSV1 and HIV-1 were performed in monocyte-derived human macrophages (hMDMs). The production of infectious HIV-1 and HSV-1 was monitored 48 h post-coinfection. Additionally, mRNA and protein expression levels of interferon-stimulated genes (ISGs) were evaluated in both HIV-1-HSV1 coinfections and HSV1 mono-infections. The HSV1 coinfection increasing the HIV-1 productive replication, following of downregulation of interferon-alpha (IFN-α) and interferon-induced transmembrane protein 3 (IFITM3) expression in hMDMs. Acyclovir treatment, in a dose-dependent manner, mitigated HSV1's ability to decrease IFITM3 levels. Knockdown of HSV1 Us11 and virion host shutoff (VHS) genes reactivated the IFN pathway, evidenced by restored IFITM3 expression and activation of eIF2-α and PKR. This knockdown also returned HIV-1 replication to baseline levels. Our data suggested that HSV1 increases HIV-1 replication in human macrophages is associated with the downregulating interferon pathways and ISGs expression.

Anti-herpes activity of the total alkaloid fraction from the branches of Fusaea longifolia (Annonaceae)

ABSTRACT The herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) occur worldwide. Infections caused by these viruses have great public health importance due to the growing resistance to the first-choice drug, acyclovir, especially in immunosuppressed patients. Alkaloids derived from species of Annonaceae have been reported as antiviral agents against HSV and others viruses. Within this context, we evaluated the antiviral activity of the total alkaloid fraction (TAF) extracted from the branches of Fusaea longifolia (Aubl.) Saff. (Annonaceae), a species native to the Amazon region, against the HSV-1 and HSV-2 viruses. The antiviral activity was evaluated through the plate reduction assay and the mode of action was investigated by a set of other assays. The TAF was active against the HSV-2 strain 333 and against the HSV-1 strains KOS and 29R (acyclovir resistant), with selectivity index values (SI = 50% cytotoxic concentration/50% effective concentration) of 5, 4 and 3, respectively. In the preliminary study of the anti-HSV-2 mode of action, TAF showed viral inhibitory effects if added up to 12 h post-infection, had virucidal activity and did not present viral inhibition in pre-treatment. Our results showed that the TAF exhibited anti-HSV activity. Regarding HSV-2, TAF acted after the viral infection and had virucidal activity. A mass spectrometry analysis revealed the presence of nine alkaloids in the TAF that had previously been reported for Annonaceae, including liriodenine, lysicamine and isoboldine, which have been described as potential anti-HSV-1 agents.

Response to HIV-1 gp160-carrying recombinant virus HSV-1 and HIV-1 VLP combined vaccine in BALB/c mice.

Human immunodeficiency virus (HIV) induced AIDS causes a large number of infections and deaths worldwide every year, still no vaccines are available to prevent infection. Recombinant herpes simplex virus type 1 (HSV-1) vector-based vaccines coding the target proteins of other pathogens have been widely used for disease control. Here, a recombinant virus with HIV-1 gp160 gene integration into the internal reverse (IR) region-deleted HSV-1 vector (HSV-BAC), was obtained by bacterial artificial chromosome (BAC) technology, and its immunogenicity investigated in BALB/c mice. The result showed similar replication ability of the HSV-BAC-based recombinant virus and wild type. Furthermore, humoral and cellular immune response showed superiority of intraperitoneal (IP) administration, compared to intranasally (IN), subcutaneous (SC) and intramuscularly (IM), that evidenced by production of significant antibody and T cell responses. More importantly, in a prime-boost combination study murine model, the recombinant viruses prime followed by HIV-1 VLP boost induced stronger and broader immune responses than single virus or protein vaccination in a similar vaccination regimen. Antibody production was sufficient with huge potential for viral clearance, along with efficient T-cell activation, which were evaluated by the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC). Overall, these findings expose the value of combining different vaccine vectors and modalities to improve immunogenicity and breadth against different HIV-1 antigens.

Primary Herpetic Gingivostomatitis.

An 82-year-old female presented with painful oral lesions associated with fever and dysphagia since 3 days. Examination revealed multiple grouped tender white to yellow plaques and few discrete vesicles on an erythematous base over the tongue and palate [Figure 1] with tender cervical lymphadenopathy. Tzanck smear from the vesicles showed multinucleate epidermal giant cells [Figure 2]. A diagnosis of primary herpetic gingivostomatitis was made and the lesions resolved following treatment with oral valacyclovir 1 g twice daily for 7 days [Figure 3]. Herpetic gingivostomatitis is typically caused by Herpes simplex virus type-1 (HSV-1), but cases caused by HSV-2 have also been reported. HSV-1 spreads by direct contact, via droplets, or by sexual contact. Primary herpetic gingivostomatitis usually presents with yellow to white plaques and vesicles which rupture and coalesce to form ulcers over the buccal mucosa, tongue, palate, gingiva, and the lips. Severe cases can have associated fever, malaise, myalgia, and local lymphadenopathy. Following the primary episode, the virus being neurotropic may become latent in the peripheral nerves and ganglions, with subsequent reactivations causing recurrent infections in around one-thirds of patients. Recurrent herpes simplex infections can present as localized vesicles commonly on the vermillion border of the lips, perioral skin, or the hard palate.[1] The diagnosis of herpes gingivostomatitis is primarily clinical. The differential diagnoses include aphthous stomatitis, oral candidiasis, herpangina, Behcet disease, erythema multiforme, Steven–Johnson syndrome, hand, foot and mouth disease and immunobullous disorders.[2] Tzanck smear from vesicles demonstrating viral cytopathic changes can support the diagnosis; however, it cannot distinguish between HSV-1, HSV-2, or varicella zoster virus. Crusted or ulcerative lesions are diagnosed by more accurate tests like direct fluorescent antibody test, polymerase chain reaction, and viral cultures. Serological tests are useful but not generally indicated for acute infections; also many people exposed to HSV may demonstrate asymptomatic seroconversion.[12]Figure 1: Multiple grouped tender white to yellow plaques and discrete vesicles on an erythematous base over the tongue and palateFigure 2: Tzanck smear showing multinucleate epidermal giant cellFigure 3: Complete resolution post treatmentMost cases of herpes gingivostomatitis are self-resolving, requiring only supportive measures. Severe cases may require systemic antiviral therapy – oral Acyclovir 15 mg/kg five times a day; or valacyclovir, 1g twice daily; or famciclovir, 500 mg twice daily; for 5 to 7 days. In case of frequent recurrences, prophylactic suppressive acyclovir (400 mg twice daily), valacyclovir (500 mg orally once daily), or famciclovir (250 mg twice daily) may be considered.[23] Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Antiviral Activity of Oridonin Against Herpes Simplex Virus Type 1.

In search of new potent treatment of herpes simplex keratitis (HSK), inhibitory effect of oridonin (Ori) on herpes simplex virus type 1 (HSV-1) was validated by experiments. For evaluating inhibitory effect of oridonin on herpes simplex virus type 1, a series of in-vivo and in-vitro studies were carried out. Mouse HSV-1 infection model was used in the in-vivo experiments. Experimental mice were classified infivedifferentgroups: Mock (mock-infected), HSV-1+ DMSO, HSV-1+ Ori, HSV-1+ ACV, combined Ori and ACV+HSV-1. Corneas of Mock, HSV-1+ DMSO, HSV-1+ Ori group were sent formRNA-sequencing after 3 days post infection (dpi). The expression of virus and host-related genes was evaluated by quantitative real-time polymerase chain reaction (qPCR). Vero cells HSV-1 infection models were used in the in-vitro experiments. The application of ACV, Oridonin alone or a combination of both could alleviate HSV-1 severity and inhibit HSV-1 virus replication in C57BL/6 mice models. qPCR showed that compared with mock group, the expression of interleukin-6 (il-6), interleukin-1α (il-1α), and Tumor-necrosis factor-alpha (tnf-α) was up-regulated in DMSO+HSV-1 group and suppressed in other three group. Moreover, the expression of nod-like receptor protein (nlrp3), caspase 1 and interleukin-1β (il-1β) were depressed in the oridonin-treated group. Oridonin significantly inhibits HSV-1 replication, HSV-1 related gene expression, and the production of progeny HSV-1 viruses in vitro. Besides, oridonin affect the replication phase but not HSV-1 entry or penetration and cannot inactivate HSV-1. Oridonin alleviates herpes simplex keratitis infection in mouse, which may be attributed to inhibition of the NLRP3-inflammasome-IL-1β pathway. Our study illustrates that Oridonin has potential promise for application in treating HSK and other diseases caused by HSV-1 infection.

Antiviral Activity of Syzygium polyanthum Extract against Herpes Simplex Virus-Type 1 (HSV-1)

Herpes simplex virus type 1 (HSV-1) is part of the alpha subfamily of human herpesviruses. Several antiviral medications can be prescribed for treating HSV-1 which is usually first line antiviral agents such as acyclovir (ACV). But there are several cases that have been reported on ACV resistance to HSV, thus, researchers tend to develop the antiviral agent from plants extract. Syzygium polyanthum is one of the plants that has been traditionally used as a drug which is rich in bioactive compounds, including essential oils, terpenoids, tannins, and flavonoids [1]. These phytochemical compounds, which have strong antioxidant action, can aid in the inhibition of viral genome replication which disables the viral lipid envelope [2].
 
 The objective of this study is to determine the cytotoxic concentration (CC50) and antiviral activity of S. polyanthum methanolic extract. Cytotoxicity was performed using the method described by [3]. The absorbance readings were analysed using microplate reader (Infinite M200 Pro). The 50% cytotoxicity concentration (CC50) was defined as the sample concentration that able to reduce 50% of cell viability compared to the untreated cells. Antiviral activity was performed by using plaque reduction assay with three different treatments [3] which are post- treatment, pre-treatment and virucidal assay.
 
 Based on Figure 1, cytotoxicity screening against Vero cells using MTT assay showed that the CC50 values for extract was 0.135 mg/mL which considered not in the range of cytotoxic compounds. Any CC50 or IC50 of a substance less than 4 µg/mL was considered an active cytotoxic effect [4].
 Based on Figure 2, post treatment assay showed the most anti-HSV-1 activity of S. polyanthum extract which the EC50 and SI value were 0.015mg/mL and 9.03, respectively. The extract showed to be slightly effective in reducing HSV-1 replication after 2-hour incubation which might be due to the inhibition in the early phase of the attachment. For pre-treatment, EC50 and SI values were 0.019 mg/mL and 7.12, which the extract showed to be moderately effective in inhibiting the attachment of HSV-1 before the replication can occur. This might occur as Vero cells bind to the extract that could interfere with the cell membrane's glycoprotein receptors which prevented HSV-1 from attaching to the cell surface [5]. The EC50 and SI value for virucidal assay was 0.018 mg/mL and 7.28 as the result showed moderately effective inhibited the viral replication. S. polyanthum has been shown to directly inactivate HSV-1 virions within 30 minutes of exposure in this study. An SI value greater than 10 (SI>10) indicates that any antimicrobial compound has the potency to become an antiviral treatment agent [6].
 In conclusion, the findings of this study demonstrate that S. polyanthum extract seems to have moderate antiviral effects and non-toxic to Vero cells.

Anti-Herpes Simplex Virus Type-1 Activity of Kyllinga nemoralis Roots Aqueous Extract

Human Herpesvirus 1 known as herpes simplex virus type 1 (HSV-1) is belonged to the Herpesviridae family, Alphaherpesvirinae subfamily, and Simplexvirus genus. Kyllinga nemoralis, known as whitehead spike sedge, white kyllinga, white globose spike, or poverty grass, is a monocotyledonous flowering graminoid similar to grasses and rushes. Acyclovir (ACV) did not eradicate HSV-1 infection as the drug only focuses on inhibiting the production of new viral genomes by interfering with viral DNA synthesis. This study aimed to identify the cytotoxic concentration (CC50) of K. nemoralis roots aqueous extract and evaluate its antiviral activities against HSV-1. 
 
 Cytotoxicity assay involved the incubation of Vero cells with 10 concentrations of extract ranging between 0.002 mg/mL and 1.000 mg/mL. MTT solution was added and absorbance was measured at 540 nm [1]. The CC50 of extract was determined from the graph of cell viability (%) against extract concentrations. Antiviral assays of post-treatment, pre-treatment and virucidal tests determined the mode of antiviral activity. The 50 % effective concentration (EC50) of extract was derived from the graph of plaque inhibition (%) against concentrations. Selectivity index (SI) was evaluated as the ratio of CC50 to EC50 [2]. 
 
 
 Post-treatment assay: Cells were inoculated with HSV-1 for 2 hours. Extract with (2% DMEM and 1% MCS) was added into cells and incubated for 48 hours. 
 Pre-treatment assay: Cells were treated with extract in 5% DMEM for 24 hours and inoculated with HSV-1 for 1 hour 30 minutes. Then, cells were overlaid with (2% DMEM and 1% MCS) and incubated for 48 hours. 
 Virucidal assay: HSV-1 was incubated with extract for 30 minutes. Then, virus-extract suspension was inoculated into cells and overlaid with 2% DMEM and 1% MCS for 48 hours. 
 
 
 After 48 hours incubation, cells were stained with crystal violet solution and incubated at room temperature for 30 minutes. The plaques were counted and percentage of plaque inhibition (%) was calculated. 
 The CC50 revealed aqueous extract concentration was able to kill at 50% of cell population. Post-treatment assay showed that 0.062 mg/mL of the extract inhibited intracellular viral replication. Pre-treatment study indicated that 0.02 mg/mL of extract produced protective effects towards cells. Finally, virucidal assay demonstrated that 0.222 mg/mL of extract initiated extracellular interaction with virus in inhibiting infection [3] as shown in Table 1. 
 The cytotoxicity study showed that CC50, 0.72 mg/mL was toxic on cells. Therefore, lower concentrations between 0.016 mg/mL to 0.5 mg/mL, that were least or non-toxic to the cells were utilized in the subsequent antiviral screenings [4]. The extract showed good inhibition of intracellular virus replication with an EC50 of 0.062 mg/mL and SI more than 10 which is 11.61. Similarly, in pre-treatment investigation, the extract produced excellent antiviral activity with an EC50 of 0.02 mg/mL and corresponding SI of 36.00. This finding highlight that the extract is most effective when administered as pre-treatment. Antiviral activity exhibited by roots extract probably results from the inhibition of host cell glycoprotein receptors or due to prevention of binding between HSV-1 virions and host cells [5]. Virucidal assay revealed a weak extracellular interaction between HSV-1 and extract. It was supported by the low SI, 3.24 as shown in Figure 1.1. The finding suggests that 30 minutes of virus-extract exposure could be insufficient for roots extract to show an effective inhibition of virus [2]. In conclusion, the present study revealed that K. nemoralis roots aqueous extract inhibits in-vitro productive HSV-1 infection through three different modes that are effective prevention of intracellular viral replication, excellent interaction with host cell surface to inhibit HSV-1 infection and moderate direct destruction the virus particles.

Antiviral Activity of Arnebia Euchroma and Amniotic Membrane against HSV-1, Rotavirus, and Influenza Virus

Background and Aims: Currently, there are antiviral chemicals used to treat viral infections accompanied by limitations such as high levels of toxicity and adverse effects in humans, the emergence of drug-resistant viral strains, low numbers, and limited diversity. Therefore, it is necessary to evaluate new photochemical to obtain new therapeutic methods. The present study was conducted to evaluate the antiviral activity of Arnebia Euchroma (A. Euchroma) extract, amniotic membrane, a mixture of A. euchroma extract and amniotic membrane and its carrier (comprised of Sesam and ostrich oil) against three different viruses, including Herpes simplex virus type 1 (HSV-1), influenza A, and rotavirus in-vitro.
 Materials and Methods: A methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the cytotoxic effect of the above compounds on Vero, MDCK, and MA-104 cell lines. After the determination of non-toxic concentrations, Tissue culture infection dose 50 (TCID50) test was performed to determine antiviral activity.
 Results: Among all the above-mentioned compounds, the combination of A. euchroma extract and amniotic membrane at the highest non-toxic concentration for the cells had the highest antiviral activity against all three viruses, leading to 1 log10 TCID50 reduction in influenza A and HSV-1 titers and 0.6 log10 TCID50 reductions in rotavirus titer when compared to the virus control.
 Conclusions: The combination of A. euchroma and amniotic membrane can be considered a new antiviral agent to treat viral infections.

Host factors associated with either VP16 or VP16-induced complex differentially affect HSV-1 lytic infection.

Herpes simplex virus type 1 (HSV-1) is an important human pathogen with neurotropism. Following lytic infection in mucosal or skin epithelium, life-long latency is established mainly in sensory neurons, which can periodically reactivate by stress, leading to recurrent disease and virus transmission. During the virus's productive infection, the tegument protein VP16, a component of HSV-1 virion, is physically associated with two cellular factors, host cell factor-1 (HCF-1), and POU domain protein Oct-1, to construct the VP16-induced complex, which is essential to stimulate immediate early (IE)-gene transcription as well as initiate the lytic programme. Apart from HCF-1 and Oct-1, VP16 also associates with a series of other host factors, making a VP16-induced regulatory switch to either activate or inactivate virus gene transcription. In addition, VP16 has effects on distinct signalling pathways via binding to various host molecules that are essentially related to innate immune responses, RNA polymerases, molecular chaperones, and virus infection-induced host shutoff. VP16 also functionally compensates for given host factors, such as PPAR-γ and ß-catenin. In this review, we provide an overview of the updated insights on the interplay between VP16 and the host factors that coordinate virus infection.

Genome-wide CRISPR screen for HSV-1 host factors reveals PAPSS1 contributes to heparan sulfate synthesis

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that causes various diseases in humans, ranging from common mucocutaneous lesions to severe life-threatening encephalitis. However, our understanding of the interaction between HSV-1 and human host factors remains incomplete. Here, to identify the host factors for HSV-1 infection, we performed a human genome-wide CRISPR screen using near-haploid HAP1 cells, in which gene knockout (KO) could be efficiently achieved. Along with several already known host factors, we identified 3′-phosphoadenosine 5′-phosphosulfate synthase 1 (PAPSS1) as a host factor for HSV-1 infection. The KO of PAPSS1 in HAP1 cells reduced heparan sulfate (HepS) expression, consequently diminishing the binding of HSV-1 and several other HepS-dependent viruses (such as HSV-2, hepatitis B virus, and a human seasonal coronavirus). Hence, our findings provide further insights into the host factor requirements for HSV-1 infection and HepS biosynthesis.

Herpes Simplex Virus 1 Induces Microglia Gasdermin D-Dependent Pyroptosis Through Activating the NLR Family Pyrin Domain Containing 3 Inflammasome

Herpes simplex virus type 1 (HSV-1) is a highly prevalent virus in humans and causes severe forms of inflammation, such as herpes simplex encephalitis (HSE). Pyroptosis is a new inflammatory cell death triggered by inflammasome and cysteine-requiring aspartate protease-1 (caspase-1) activation. Nonetheless, HSV-1 induces encephalitis, and cell death mechanisms are not understood. In this study, we confirmed for the first time that the DNA virus HSV-1 triggers Gasdermin D-dependent pyroptosis by activating NLR family pyrin domain containing 3 (NLRP3) inflammasomes in mouse microglia, leading to mature IL-1β production and active caspase-1 (p10) release. Inhibition of microglial NLRP3 inflammasome activation suppressed HSV-1-induced Gasdermin D-dependent pyroptosis. In addition, NLRP3 and IL-1β expression levels were significantly increased in the mouse model of herpes simplex encephalitis compared with normal mice without viral infection. Collectively, our data revealed that the activation of inflammasomes and GSDMD-dependent pyroptosis is the mechanism of HSV-1 inducing inflammation and provides treatment targets for viral inflammation.

Characterizing the role of Tupaia DNA damage inducible transcript 3 (DDIT3) gene in viral infections

DNA damage inducible transcript 3 (DDIT3, also known as CHOP) belongs to the CCAAT/enhancer-binding protein (C/EBP) family and plays an essential role in endoplasmic reticulum stress. Here, we characterized the potential role of the Chinese tree shrew (Tupaia belangeri chinensis) DDIT3 (tDDIT3) in viral infections. The tDDIT3 protein is highly conserved and has a species-specific insertion of the SQSS repeat upstream of the C-terminal basic-leucine zipper (bZIP) domain. Phylogenetic analysis of DDIT3 protein sequences of tree shrew and related mammals indicated a closer genetic affinity between tree shrew and primates than between tree shrew and rodents. Three positively selected sites (PSSs: Glu83, Pro93, and Ser172) were identified in tDDIT3 based on the branch-site model. Expression analysis of tDDIT3 showed a constitutively expressed level in different tissues and a significantly increased level in tree shrew cells upon herpes simplex virus type 1 (HSV-1) and Newcastle disease virus (NDV) infections. Overexpression of tDDIT3 significantly increased the production of HSV-1 and vesicular stomatitis virus (VSV) in tree shrew primary renal cells (TSPRCs), whereas tDDIT3 knockout in tree shrew stable cell line (TSR6 cells) had an inhibitory effect on virus production. The enhanced effect on viral infection by tDDIT3 was not associated with the three PSSs. Mechanistically, tDDIT3 overexpression inhibited type I IFN signaling. tDDIT3 interacted with tMAVS through CARD and PRR domains, but not with other immune-related factors such as tMDA5, tSTING and tTBK1. Collectively, our results revealed tDDIT3 as a negative regulator for virus infection.

Viperin has species-specific roles in response to herpes simplex virus infection.

Viperin is a gene with a broad spectrum of antiviral functions and various mechanisms of action. The role of viperin in herpes simplex virus type 1 (HSV-1) infection is unclear, with conflicting data in the literature that is derived from a single human cell type. We have addressed this gap by investigating viperin during HSV-1 infection in several cell types, spanning species and including immortalized, non-immortalized and primary cells. We demonstrate that viperin upregulation by HSV-1 infection is cell-type-specific, with mouse cells typically showing greater increases compared with those of human origin. Further, overexpression and knockout of mouse, but not human viperin significantly impedes and increases HSV-1 replication, respectively. In primary mouse fibroblasts, viperin upregulation by infection requires viral gene transcription and occurs in a predominantly IFN-independent manner. Further we identify the N-terminal domain of viperin as being required for the anti-HSV-1 activity. Interestingly, this is the region of viperin that differs most between mouse and human, which may explain the apparent species-specific activity against HSV-1. Finally, we show that HSV-1 virion host shutoff (vhs) protein is a key viral factor that antagonises viperin in mouse cells. We conclude that viperin can be upregulated by HSV-1 in mouse and human cells, and that mouse viperin has anti-HSV-1 activity.

Abstract 1586: Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic tumor models

Abstract Oncolytic virotherapy is an emerging treatment modality that may hold promise as a therapeutic option for cancer patients who do not respond to checkpoint inhibitors. Oncolytic viruses preferentially replicate in tumor cells, offer the ability to express transgenes and have been shown to modify the local and distant tumor microenvironments from immunosuppressive to immunostimulatory. We generated HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12, an oncolytic virus based on a modified herpes simplex virus type 1 (HSV-1) designed to selectively replicate in tumors and produce murine FMS-like tyrosine kinase 3 ligand (mFLT3L) to enhance dendritic cell expansion and T cell priming, and murine interleukin-12 (mIL12) to potentiate type I helper and cytotoxic T cell responses. We confirmed the expression and bioactivity of mFLT3L and mIL12 in vitro and evaluated the efficacy of HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 alone and in combination with anti-PD-1 or anti-4-1BB treatment in multiple syngeneic mouse tumor models. Bilateral tumor models were used and only one tumor was treated via intratumoral injection with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12. Anti-tumor responses were evaluated in the injected tumor (local response), the uninjected tumor (abscopal) and via splenic immune profiling (systemic). HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 treatment alone led to tumor growth inhibition in both treated and contralateral tumors, and a significant increase in overall survival in the A20, Neuro2a and MC38 models. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with anti-PD-1 blocking antibody further improved tumor growth inhibition in both treated and contralateral tumors in the MC38 syngeneic model, and significantly increased median overall survival compared to either agent as a monotherapy. In addition, HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with an agonistic antibody targeting 4-1BB significantly increased overall survival compared to either agent as a monotherapy in the MC38 model. To understand antigen-specific anti-tumor T cell responses, MC38 tumor antigen epitopes were identified by combining exome sequencing and MHC/HLA-binding prediction algorithms. Antigen-specific T cell responses were measured in an IFN-γ ELISPOT assay using splenocytes from treated mice. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 induced and/or enhanced systemic T cell responses directed against both tumor neoantigens and tumor-associated antigens. Together, these data reveal that treatment with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 can cause direct tumor lysis and induce an adaptive, systemic T cell-mediated anti-tumor immune response that can be further enhanced by concurrent PD-1 blockade or 4-1BB agonism in multiple syngeneic mouse tumor models. Citation Format: Keegan Cooke, Walter H. Meisen, Petia Mitchell, Juan Estrada, Jinghui Zhan, Jessica Orf, Peng Li, Christina de Zafra, Jing Qing, Jason DeVoss. Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1586.

Study of the Extremely-Tolerant Brevibacterium linens AE038-8 with Antiviral Activity Against Herpes Simplex Virus Type 1.

Brevibacterium linens AE038-8 is an arsenic hyper-tolerant bacterial strain, previously isolated from well water in Tucumán, Argentina. The aim of this study was to characterize this strain regarding its resistance to different stress factors and to evaluate its antiviral activity against Herpes simplex virus type 1 (HSV-1). We found that B. linens AE038-8 was capable of tolerating high concentrations of heavy metals such as Cd(II), Cr(VI) and Cu(II). When grown in the presence of NaCl, it could tolerate up to 3M in LB25 medium. When cultivated, B. linens released to the supernatants a bioactive principle with antiviral activity against HSV-1 virus regardless growth conditions.

Antiviral activity of extracts of basidiomycetes and humic compounds substances against Human Immunodeficiency Virus (Retroviridae: Orthoretrovirinae: Lentivirus: Human immunodeficiency virus 1) and Herpes Simplex Virus (Herpesviridae: Simplexvirus: Human alphaherpesvirus 1)

One of the most urgent problem of modern medicine is the fight against the disease caused bythe Human Immunodeficiency Virus (HIV) - HIV infection. The chemical compounds have improved the situationfor infected people, but they are toxic, disrupt the metabolism and cannot eliminate the integrated virus from thebody. The emergence of resistant HIV strains makes these treatments ineffective. Often, the death of HIV-infectedpeople occurs as a result of the development of opportunistic infections caused by viruses of the Herpesviridaefamily. Therefore, the search for new therapeutic and preventive drugs that are less toxic and active against severalviruses at the same time is relevant. Basidiomycetes, higher fungi, are a source of medicinal compounds that haveantimicrobial properties, as well as antiviral ones. Humic compounds (HS) of various nature also have antiviralactivity.The aim of the study was to obtain nontoxic compounds from the basidiomycete Inonotus obliquus and humiccompounds from brown coals and to test their activity against viruses that are pathogenic to humans: HIV andHerpes Simplex Virus (HSV). The antiviral activity of melanin extracts obtained from the culture of the chaga fungusInonotus obliquus and HS from the brown coal of the Kansko-Achinsk Deposit was studied using a model of MT-4lymphoblastoid cells infected with HIV type 1 (HIV-1) strains and a monolayer culture of Vero cells infected withHSV type 1 (HSV-1) using virological and statistical research methods. It was found that all the studied compounds did not have a cytotoxic effect on cells at aconcentration of 100 mcg/ml. It was shown that extracts of basidiomycetes and HS have antiviral activity againstHIV-1 and HSV-1. EC50 (50%-effective concentration) for HIV-1 was 3.7-5.0 mcg/ml, selectivity index 28-35.Antiherpetic activity was detected at a dose of 50-100 mcg/ml. The antiviral effectiveness of melanin compoundswas established both in the «preventive» (2 hours before cell infection) and in the «therapeutic» regimen of drugadministration, both for HIV-1 and HSV-1. The presence of antiviral activity of melanin and HS in relation to theRNA-containing HIV-1 virus and DNA-containing HSV-1 virus in our study coincides with the results of a number ofauthors in relation to influenza viruses, herpes virus, HIV, hepatitis B virus, Coxsackievirus, smallpox vaccine virus,which suggests that the type of nucleic acid in the virus does not play a fundamental role in the antiviral action ofthese drugs. It is also clear that HS is effective against both enveloped and non-enveloped viruses. In general, it can be concluded that melanin and humic compounds are characterized by low toxicityin the presence of both virucidal and antiviral activity. This allows us to consider the studied compounds as thebasis for creating safe medicines that are effective against pathogens of various viral infections.